FIG. 4.

Coomassie brilliant blue-stained (top) and Western immunoblot analysis (bottom) of whole bacterial lysates from live vectors expressing ClyA-PA83 fusions from SSB-maintained plasmids carried by CVD 908-htrAssb (lanes 2 to 4) and CVD 908ssb (lanes 5 and 6). Lysates from strains carrying medium-copy-number derivatives of pSEC91S are in lanes 2, 3, and 5; lysates from strains carrying low-copy-number derivatives of pSEC10S are in lanes 4 and 6. A lysate from the conventional strain CVD 908-htrA(pSEC91-83) expressing ClyA-PA83 from a medium-copy-number kanamycin resistance plasmid is in lane 7, and 125 ng of purified PA83 is in lane 1. Total protein from approximately 10⁶ CFU was resolved in each lane. Detection of ClyA-PA83 fusions was carried out using a polyclonal goat anti-PA83 primary antibody. The values at the left are molecular masses in kilodaltons. The arrows on the right indicate the position of the ClyA-PA83 fusion protein, with an expected molecular mass of 117 kDa.